Since their introduction in 1990, aptamers have become an increasingly mainstream alternative to antibodies as affinity reagents for targets ranging from small molecules to large proteins and even whole cells. They offer important advantages over antibodies that make them attractive candidates for wide-ranging analytical and medical applications. The process of aptamer discovery begins with construction of a random oligonucleotide library that is reduced through an iterative selection process, Systematic Evolution of Ligands by Exponential Enrichment (SELEX), to a handful of sequences that exhibit high affinity to the target. There is no question that aptamers, once discovered, can live up to their promise. The limiting factor in achieving their full potential is the relatively sall number of protein targets to which aptamers have been selected despite intensive effort and substantial investments of time and money. These failures have been attributed to a number of factors including the SELEX process itself, which is notoriously laborious, non-standardized and difficult to fully automate. Yet even if these problems are solved, there remain fundamental difficulties inherent in SELEX that will continue to hinder progress in aptamer discovery. These include the quality of the combinatorial library, under-representation of certain structural motifs and inefficiencies and bias of PCR amplification. This proposal offers a paradigm shift in aptamer discovery that overcomes many of the roadblocks to aptamer selection. The proposed, genome-inspired approach reverses the selection, using specific DNA sequences from the human genome to capture proteins from natural pools such as nuclear protein extracts. This approach takes advantage of eons of biological evolution that produced genomic DNA sequences that selectively bind to proteins to perform biological functions. Specifically, the goal is to identify proteins that specifically bind to G-quadruplex (G4) sequences in breast cancer gene promoter regions. Although G4 is a highly successful aptamer motif, it is underrepresented in combinatorial libraries; those present are limited to two-tier G4 structures and are prone to inefficient PCR amplification. In contrast, there is a large diversity of multi-tiered G4 structure throughout the human genome including promoter regions of breast cancer genes. The proposed approach mines these previously untapped structures, opening the door to aptamers to new protein targets. Specific Aim 1 is to systematically evaluate the proposed reverse selection approach using genomic G4 forming sequences from the promoter regions of breast cancer-related human genes to capture proteins extracted from cell nuclei of cultured breast cancer and normal breast tissue cell lines. Specific Aim 2 is to perform chromatin immunoprecipitation (ChIP) to determine if a protein selected in vitro binds to the gene promoter regions containing the corresponding G4-forming sequence in the chromatin of live, cultured cells. The research will lead to aptamers to proteins involved in nuclear regulatory processes related to breast cancer, possibly including proteins not yet recognized to be important targets for SELEX.